Type I modular PKSs are responsible for generating the macrolide core of a diverse range of polyketide products with pharmaceutical, veterinary, and agricultural applications. The modular nature of type I PKSs have made them particularly attractive targets for enzyme bioengineering efforts, establishing a new and exciting approach to discovery and development of natural product pharmaceuticals. There is significant optimism that the creation of unnatural hybrid PKSs can enable structural modification and development of these natural products into therapeutic agents, and several strategies have been used. Understanding how individual proteins within a multi-component polyketide synthase (PKS) interact with one another to create a functional assembly line has been integral to creating engineered biosynthetic pathways. The protein-protein interfaces are thought to be largely mediated by the coiled-coil termini motifs called docking domains that enable specific pair-wise interactions for effective catalytic activity. One can envision a synthetic biology approach in utilizing a diverse range of cognate dock domain pairs for the construction of new PKS pathways. A similar type of coupling occurs between interacting modules of non-ribosomal polypeptide synthases (NRPSs). Potentially, novel PKS-NRPS hybrid pathways could be engineered by mediating protein-protein interactions through specific couplings. A key question remains whether modular PKS and NPRS proteins from phylogenetically divergent sources (e.g. actinomycetes, marine cyanobacteria, myxobacteria) can interact more productively if engaged through a strong binding interface. This question provides a compelling motivation to explore the ability of synthetic high affinity DNA binding domains (DBDs) to mediate PKS modular interactions for efficient assembly of novel polyketide natural product molecules. DBDs are ubiquitously found in biological systems including bacteria, fungi, mammals and viruses. DBD refers to an independently folded protein domain, which contains at least one motif that recognizes double- or single-stranded DNA. DBDs have a number of characteristics that make them extremely attractive for protein engineering. The noteworthy feature is their high affinity (KD <50 nM) to bind to sequence-specific double stranded DNA. Therefore, DBDs are an attractive target to replace the relatively low affinity PKS docking domains (KD ~ 50 5M). The aim of this proposal is to explore the use of DBDs as a means to establish high affinity interactions between PKS modules while maintaining efficient catalytic activity. Although much is known about DBDs, their application as artificial docking domains has not been explored. A number of features of the DBDs will likely need optimizing including the choice of DBD, the size of the domain, and the DNA sequence used to bring two distinct DBDs together. DBD fusion proteins will be tested for functionality and the ability to control interactions strictly will be examined. Finally, a number of hybrid PKS systems will be tested for their potential to generate novel polyketide molecules. The proposed work provides significant potential to unlock the modular potential of PKS and NRPS systems for the generation of new biologically active natural products. PUBLIC HEALTH RELEVANCE: The rapid rise of antibiotic resistant microbes has made the discovery and development of novel antibacterial agents a priority for national health. Polyketide natural products, such as erythromycin, have proven to be a rich source of antimicrobial bioactivity;however, due to their structural complexity, the synthesis of novel polyketides is a challenging, costly, and time consuming endeavor. The modular nature of PKS systems is an attractive feature for discovery and development of new macrolide antibiotics. The synthesis of biologically active polyketide natural product molecules is mediated by a multi-component complex comprised of proteins linked to each other in an analogous fashion to that of a passenger train, with each car representing a protein whose sequential order is dictated by the unique coupling mechanism between proteins. Each protein 'car'performs a specified modification to the polyketide molecule as it transits head-to-tail through the protein 'train'. The order of the protein 'cars'dictates the final size and shape of the polyketide molecule. By re-engineering the coupling mechanism between proteins we plan to rearrange the order of the assembly proteins within the protein 'train'resulting in new, polyketide molecules with diverse biological activities.